The rapid developments in recombinant DNA methodology have allowed the production of polypeptides, proteins, and their analogs in unlimited quantities in a very short period of time. These developments have created a need to handle purification of these proteins from complex mixtures in highly efficient and predictable manners.
Immobilized metal ion affinity chromatography (IMAC) has been used extensively to purify proteins containing exposed histidine and cysteine side chains. See Porath, J. et al., Nature, 258:598-599 (1975). Chelating agents selective for specific metal ions are coupled to a solid matrix, such as agarose beads or silica. For example, an active derivative of the chelating moiety nitrilotriacetic acid (NTA; N,N-bis[carboxymethyl]glycine) can be coupled to resins or solid matrices for use with metal ions having coordination numbers of six (e.g., Ni2+), other chelating moieties include iminodiacetic acid (IDA) derivatives and tris (carboxymethyl) ethylenediamine. Sharma (U.S. Pat. No. 5,594,115) describes the use of Ni2+ to purify histidine tagged peptides.
According to the method of the invention, polypeptides and/or proteins can be isolated and subsequently purified from an aqueous solution containing same by contacting the aqueous solution with a composition suitable for removing a peptide or protein of interest contained in said solution, then recovering the peptide or protein. Compositions useful for carrying out the methods of the invention comprise an ion-binding ligand bound to a membrane loaded with a metallic ion having an affinity for polypeptides/proteins contained in the aqueous solution. The membrane ligand combination is represented by the formula:
M-B-L-X
wherein M is a membrane or composite membrane derivatized to have a hydrophilic surface and containing polar functional groups; L is a ligand having an affinity for metallic ions and containing a functional group reactive with an activated polar group from the membrane; B is a covalent linkage formed by the reaction between the activated polar group and the functional group of the ligand L; and X is a metallic ion chelated to L and has an affinity for polypeptides/proteins. The metallic ion is selected from the group consisting of cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn) or combinations thereof.
Separation of peptides and/or proteins using membrane based metal ion affinity chromatography allows efficient and rapid removal of material, at high throughput, due to the high surface area provided by the membranes. Another advantage of membrane based separations is that equilibrium can be reached quickly. Further, micron-sized samples can be purified using an array of membranes, e.g., 96-well, 384-well and 1536-well separation devices, for automation purposes.